The present invention relates to recombinant DNA which encodes the AatII restriction endonuclease and modification methylase and the Alul restriction endonuclease and modification methylase, and the production of these enzymes from the recombinant DNA, as well as to related methods for overexpressing endonucleases.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over one hundred and fifty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date.
Bacteria tend to possess at most, only a small number of restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, synthesizes three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences TTTAAA, PuGGNCCPy and CACNNNGTG respectively. Escherichia coli RY 13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC.
While not wishing to be bound by theory, it is thought that in nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by cleaving an invading foreign DNA molecule each time that the recognition sequence occurs. The cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific endonucleases.
A second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e. populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
Type II restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Molec. gen. Genet, 178:717-719 (1980); HhaII: Mann et al., Gene, 3:97-112 (1978); PstI: Walder et al., Proc. Nat. Acad. Sci., 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acid. Res., 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci, USA, 80:402-406 (1983); Theriault and Roy, Gene, 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol., 164:501-509 (1985)).
A third approach, and one that is being used to clone a growing number of systems involves selection for an active methylase gene (see, e.g., U.S. Pat. No. 5,200,333 to Wilson and BsuRI: Kiss et al., Nucl. Acid. Res., 13:6403-6421 (1985)). Since restriction and modification genes are often closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead often yields only the methylase gene (BspRI: Szomolanyi et al., Gene, 10:219-225 (1980); Bcn I: Janulaitis et al., Gene, 20:197-204 (1982); Bsu RI: Kiss and Baldauf, Gene, 21:111-119 (1983); and Msp I: Walder et al., J. Biol. Chem., 258:1235-1241 (1983)).
In some systems the cloning problem may lie in trying to introduce the endonuclease gene into a host not already protected by modification. If the methylase gene and endonuclease gene are introduced on a common DNA fragment, the methylase gene must modify or protect the host before the endonuclease gene cleaves the host's genome.
Another obstacle to cloning these systems in E. coli was discovered in the process of cloning diverse methylases. Many E. coli strains (including those normally used in cloning) have systems that resist the introduction of DNA containing cytosine methylation. (Raleigh and Wilson, Proc. Natl. Acad. Sci. USA, 83:9070-9074 (1986) ). Therefore, it is also necessary to carefully consider which E. coli strain(s) to use for cloning.
When foreign restriction modification systems are cloned and introduced into E. coli, sometimes the endonuclease yield is very low compared to the native endonuclease-producing strain probably due to inefficient transcription or translation of the gene in E. coli. In some cases, E. coli cells carrying a cloned restriction modification system grow poorly probably due to insufficient methylation protection and unregulated constitutive expression of the restriction endonuclease gene. Therefore, a tightly regulated expression system would be desirable in order to express toxic genes such as restriction endonuclease genes in E. coli.
It therefore would be desirable to have an expression system which will produce a minimal level of endonuclease under noninduced conditions and produce large amounts of the desired endonuclease upon induction. In this way foreign restriction modification systems can be stably maintained in E. coli hosts.
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing genes in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification and would provide the means for production in commercially useful amounts.